JP2023026238A - Fluid processing cartridge, as well as nucleic acid recovery and amplification method - Google Patents

Abstract

To provide a cartridge that enables implementation of amplification to detection of a nucleic acid recovered from an analyte within the same device, and enables making the device easy in transportation means of a fluid more compact, as well as to provide a nucleic acid recovery and amplification method.SOLUTION: A fluid processing cartridge has: a plurality of fluid storage tanks; flow channel switch means; and a flow channel between each liquid storage tank and the flow channel switch means. The flow channel switch means includes: a rotor; and a cylinder in which the rotor is slidable while keeping a tight contact state between an outer periphery surface of the rotor and an inner periphery surface thereof. The rotor has, in the outer periphery surface, at least one group making a plurality of flow channel ports the cylinder has communicatable by rotation or parallel movement of the rotor, and/or the rotor has at least one through-hole making the plurality of flow channel ports the cylinder has communicatable by the rotation or parallel movement of the rotor. The present invention relates to a nucleic acid recover and amplification method, using the cartridge.SELECTED DRAWING: Figure 1

Description

The present invention relates to fluid handling cartridges and nucleic acid recovery and amplification methods. More particularly, the present invention relates to fluid processing cartridges suitable for recovery, amplification and detection of nucleic acids from specimens and nucleic acid recovery and amplification methods using the cartridges.

With the spread of COVID-19, the demand for techniques for recovering, amplifying, and detecting nucleic acids in viruses to simply and reliably determine the presence or absence of virus infection is increasing day by day. Also, even after the problem of COVID-19 is overcome, there is still a need for similar technology as mankind faces various viral and bacterial diseases.

Techniques for amplifying and detecting nucleic acids have been established as methodology with the advent of the PCR method and the subsequent development of various methods other than the PCR method. It has come to be done in However, even then, nucleic acids are often isolated from biological samples such as viruses manually, and then amplified and detected using existing amplification and detection devices. However, with the spread of COVID-19, it is clear that existing methods and devices cannot be used to deal with serious cases by bringing specimens to public health centers and testing companies. At present, it is not possible to reliably determine the presence or absence of virus infection by processing a large amount of specimens anywhere in a short time and simply.

Patent Literature 1 discloses a small portable amplification and detection device that can more easily amplify and detect nucleic acids.

WO2018/123837 WO2002/023776

The apparatus described in Patent Document 1 is an apparatus for separately recovering nucleic acids from a sample, and then amplifying and detecting the recovered nucleic acids. However, in the method using this device, it is necessary to collect the nucleic acid separately from the specimen, and the virus cannot be detected from the specimen all at once.

On the other hand, the present inventors have planned to recover nucleic acids from a sample, amplify the recovered nucleic acid, and detect the amplified nucleic acid using a single device, using the sample as it is without pretreatment. . In this device, the steps of recovering, amplifying, and detecting nucleic acids are performed sequentially and automatically in chambers connected by microchannels, and in consideration of the high nucleic acid recovery efficiency, a carrier such as a filter is used. assume to do.

An apparatus for recovering nucleic acids using a carrier is known (Patent Document 2). The device has a plurality of chambers containing a plurality of reagent solutions and the like and a mechanism for moving fluid between the chambers. The inter-chamber fluid transfer mechanism comprises a tubular body having a fluid transfer chamber therein, a piston that moves up and down within the tubular body to depressurize or pressurize the interior of the tubular body, and another at the bottom of the fluid transfer chamber. It has a switchable flow path that connects with the chamber, and utilizes reduced pressure or increased pressure inside the tubular body to recover nucleic acids from the specimen while moving the fluid between the fluid transfer chamber and other chambers. conduct. However, with this device, the liquid must be moved by vertical movement of the piston, which poses the problem of a complicated operating mechanism. do not have.

The problem to be solved by the present invention is to provide a cartridge that allows amplification and detection of the recovered nucleic acid to be carried out in the same device, facilitates liquid transfer means, and allows a more compact device for mounting the cartridge. to do.

The present invention is as follows.
[1]
A fluid processing cartridge comprising a plurality of liquid storage tanks, channel switching means, and channels between each liquid storage tank and the channel switching means,
The flow path switching means includes a rotor and a cylinder in which the rotor can slide while the outer peripheral surface and the inner peripheral surface of the rotor are maintained in close contact,
The rotor has at least one groove on its outer peripheral surface that enables communication between a plurality of flow passage ports of the cylinder by rotation or translation of the rotor, and/or The rotor has, by rotation or translation of the rotor, the cylinder. having at least one through-hole that allows communication of a plurality of flow channel ports;
Said cartridge.
[2]
The cartridge according to [1], wherein the groove has a through-hole opening from the inside of the rotor.
[3]
The rotor is a solid cylinder or has a cylindrical outer wall having a cavity inside, and the through hole is a hole passing through the solid cylinder or a hole passing through the cylindrical outer wall, A cartridge according to [1] or [2].
[4]
The cartridge according to any one of [1] to [3], wherein the plurality of liquid storage tanks, channels and cylinders are integrated, and the rotor is insertable through the opening of the cylinder.
[5]
Any one of [1] to [4], wherein both ends of the rotor are openings, and the rotor has an internal space from each opening and an internal structure separating two or more internal spaces. 1. The cartridge according to item 1.
[6]
The cylinder has a closed end on the inner side of the cylinder, or has a nozzle extending from the inner side of the cylinder to the inner space of the rotor, is closed except for the nozzle, has an opening at the tip of the nozzle, and the nozzle is the cylinder The cartridge according to any one of [1] to [5], which is capable of communicating with the channel of
[7]
The cartridge according to any one of [1] to [6], wherein the fluid treatment cartridge has a carrier in the channel switching means or in the middle of the channel between the liquid storage tank and the channel switching means.
[8]
When both ends of the rotor are openings and the rotor has an internal structure with an internal space from each opening and separating two or more internal spaces,
The cartridge according to [7], wherein the carrier in the channel switching means is included in the internal structure inside the rotor.
[9]
When both ends of the rotor are openings and the rotor has an internal structure with an internal space from each opening and separating two or more internal spaces,
The cartridge according to any one of [5] to [8], wherein the internal structure includes a liquid receiving portion on the outside of the cylinder, and the liquid receiving portion has a discharge passage that can communicate with the outside of the rotor.
[10]
The cartridge of [9], wherein the liquid receiver is funnel-shaped.
[11]
The fluid treatment cartridge according to any one of [1] to [10], further comprising a waste liquid recovery tank connected to the channel switching means via a channel, and having an exhaust port in the waste liquid recovery tank. Cartridge as described.
[12]
The cartridge according to [11], wherein when the fluid processing cartridge is used, the exhaust port is located above the waste liquid tank inlet in the waste liquid collection tank.
[13]
The cartridge according to [11] or [12], wherein when the fluid processing cartridge is used, the liquid in the liquid storage tank and the flow path is circulated and exhausted by the negative pressure generated by the exhaust from the exhaust port.
[14]
The cartridge according to any one of [1] to [13], wherein the fluid processing cartridge further includes a reaction tank connected to the channel switching means via a channel.
[15]
At least some of the plurality of liquid storage tanks and the reaction tank have ventilation holes or air passages leading to the ventilation holes, and when the fluid processing cartridge is used, the communication port to the ventilation holes of the liquid storage tanks is the liquid storage tank. The cartridge according to any one of [1] to [14], located above.
[16]
The cartridge according to any one of [1] to [15], wherein the positional relationship between the rotor and the cylinder in the channel switching means has at least a position where the reaction vessel is closed.
[17]
In the channel switching means, the positional relationship between the rotor and the cylinder has a structure and position of grooves and through holes in which all channels are disconnected via the channel switching means, [1] to [16] A cartridge according to any one of the preceding claims.
[18]
The cartridge according to any one of [1] to [17], wherein the rotor has one or more grooves and one or more through holes.
[19]
The plurality of liquid storage tanks are one or two or more sample suspension tanks, one or two or more washing liquid tanks, one or two or more eluent tanks, one or two or more mixing tanks, The cartridge according to any one of [1] to [18], wherein the fluid processing cartridge further comprises one or more waste reservoirs and one or more reaction reservoirs.
[20]
The cartridge according to any one of [1] to [19], wherein the cartridge is for recovering, amplifying and/or detecting nucleic acid from a specimen, and nucleic acid amplification is performed in a reaction vessel.
[21]
A method comprising recovering and amplifying a nucleic acid using the cartridge according to any one of [1] to [20].
[22]
When nucleic acid amplification is carried out in a reaction vessel, the rotor and cylinder of the channel switching means take at least a position in which the reaction vessel is closed, or a position in which all channels are disconnected, [21 ] The method described in .
[23]
The method of [21] or [22], further comprising the step of optically detecting, electrically detecting, or detecting by surface plasmon resonance the amplified nucleic acid after the nucleic acid amplification operation.

According to the present invention, it is possible to provide a cartridge in which amplification and detection of the recovered nucleic acid can be carried out in the same device, a liquid transfer means is easy, and the device in which the cartridge is mounted can be made more compact. can. By using this cartridge, the target nucleic acid can be detected more reliably.

1 shows a schematic diagram of an example of a fluid treatment cartridge of the present invention; FIG. FIG. 4 is a schematic horizontal cross-sectional view of a channel switching means included in an example of the fluid processing cartridge of the present invention; FIG. 4 shows a schematic vertical cross-sectional view of a channel switching means included in an example of the fluid processing cartridge of the present invention. 1 shows a vertical cross-sectional schematic illustration of the cylinder and rotor of one example of a fluid treatment cartridge of the present invention; FIG. FIG. 4 is a schematic horizontal cross-sectional view of a channel switching means included in an example of the fluid processing cartridge of the present invention; FIG. 4 is a schematic horizontal cross-sectional view of a channel switching means included in an example of the fluid processing cartridge of the present invention; FIG. 4 is a schematic horizontal cross-sectional view of a channel switching means included in an example of the fluid processing cartridge of the present invention; 1 shows a vertical cross-sectional schematic illustration of the cylinder and rotor of one example of a fluid treatment cartridge of the present invention; FIG. 1 shows a vertical cross-sectional schematic illustration of the cylinder and rotor of one example of a fluid treatment cartridge of the present invention; FIG. 1 shows a vertical cross-sectional schematic illustration of the cylinder and rotor of one example of a fluid treatment cartridge of the present invention; FIG.

<Fluid treatment cartridge>
The present invention relates to a fluid processing cartridge, which has a plurality of liquid storage tanks, channel switching means, and channels between each liquid storage tank and the channel switching means. The flow path switching means includes a rotor and a cylinder in which the rotor can slide while the outer peripheral surface and the inner peripheral surface of the rotor are kept in close contact with each other. The rotor can have at least one groove on its outer peripheral surface that can communicate with a plurality of flow passage ports of the cylinder by rotating or translating the rotor. The rotor can have at least one through-hole that can communicate with a plurality of flow channel ports of the cylinder by rotating or translating the rotor.

A fluid processing cartridge according to the present invention will now be described with reference to FIG. FIG. 1 is a schematic diagram of an example of the fluid processing cartridge of the present invention.

In FIG. 1, 10 is a flow path switching means, 20 to 25 are liquid storage tanks, and 50 to 57 are flow paths between each liquid storage tank and the flow path switching means. Reference numerals 58 and 59 denote channels between the waste liquid recovery tank and the channel switching means, and 60 denotes a channel between the reaction tank and the channel switching means. In FIG. 1, 6 liquid storage tanks numbered 20 to 25 are shown as an example, but the number of liquid storage tanks is not particularly limited as long as it is 2 or more, 2, 3, 4, 5, 6, 7, 2, 3, 4, 5, 6, 7, There may be 8, 9, 10 or more. Although 50 to 60 flow paths are shown in FIG. 1, the number of flow paths can be appropriately selected according to the number of liquid storage tanks, waste liquid recovery tanks, and reaction tanks. In the cartridge of the present invention, the plurality of liquid storage tanks, channels and cylinder can be integrated, and the rotor can be inserted from the opening side of the cylinder. For example, the opening of the cylinder is arranged at the bottom of the cartridge (the surface on the lower side in the longitudinal direction of the channel switching means 10). The arrangement of the rotor and cylinder will be described later.

The flow path switching means 10 includes a rotor and a cylinder in which the rotor can slide while the outer peripheral surface and the inner peripheral surface of the rotor are kept in close contact with each other. The rotor and cylinder being slidable means that the rotor is rotatable in the cylinder and/or the rotor is translatable in the cylinder. The flow channel switching means consisting of a rotor and a cylinder has a vertically long cylindrical shape, and the flow channel is connected to the side peripheral surface. It can also be placed in oblique positions at various angles. In order to maintain close contact between the outer circumference of the rotor and the inner circumference of the cylinder, the outer diameter of the rotor and the inner diameter of the cylinder should be approximately equal, and the outer circumference of the rotor and the inner circumference of the cylinder should be smooth enough to maintain close contact. Preferably. Also, during this time, various lubricating oils can be used so that the cylinder and rotor can be brought into close contact with each other and can be rotated and pulled out.

The rotor can be a solid cylinder or have a cylindrical outer wall with an internal cavity. The rotor can have at least one groove on its outer peripheral surface that can communicate with a plurality of flow passage ports of the cylinder depending on the rotational position of the rotor. The grooves can be oriented horizontally or oriented vertically. Although it can be provided with an inclination, considering the operability of the flow path switching means 10, it is preferable to provide it in the horizontal direction or the vertical direction. The length and depth of the groove can be appropriately determined in consideration of the intervals and dimensions of the plurality of channel openings of the cylinder.

FIG. 2 shows a horizontal cross-sectional schematic illustration of a rotor and a cylinder having a cylindrical outer wall with a cavity inside. An example in which grooves are provided horizontally on the outer peripheral surface of the rotor is shown. In FIG. A, both ends of groove A connect channel 1 and channel 2 . FIG. B shows a state in which the rotor is rotated, both ends of groove A are disconnected from the opening of channel 1 and the opening of channel 2, and communication between channel 1 and channel 2 is closed. . In Figure A, groove A can be used to allow liquid or gas to flow between channel 1 and channel 2 . In the state of FIG. B, the communication between channel 1 and channel 2 is closed and no liquid or gas flow using groove A is possible. In order to break the connection between the two flow paths, the groove should be in non-contact with at least one of the two flow paths.

FIG. 3 shows a schematic illustration of a vertical cross section of the rotor and cylinder. An example in which the grooves are provided in a direction perpendicular to the outer peripheral surface of the rotor is shown. In FIG. A, both ends of the groove B connect the channel 3 and the channel 4 . FIG. B shows a state in which the rotor is rotated, both ends of groove B are disconnected from the opening of channel 3 and the opening of channel 4, and communication between channel 3 and channel 4 is closed. . Groove B can be used in the state of FIG. In the state of FIG. B, the communication between channels 3 and 4 is closed, and no liquid or gas can flow through groove B. FIG.

FIG. 4 shows a schematic illustration of a vertical cross section of the rotor and cylinder. The rotor having a cylindrical outer wall with an internal cavity, shown in FIG. 4, is cylindrical and open at both ends. Within the cylinder, the rotor has openings at the cylinder-inside end and the cylinder-outside end, and the rotor has an interior space from each opening. The internal space of the rotor can be just a space, as shown in FIG. 3, or it can have an internal structure separating the two internal spaces, as shown in FIG. The cylinder may be closed at the cylinder inner end, as shown in FIG. 3, or may have nozzles extending from the cylinder inner side into the rotor inner space, as shown in FIG. closed except for the rotor insertion opening). The tip of the nozzle is an opening and the nozzle communicates with the flow path 10 inside the cylinder. In view A of FIG. 4, channel 3 leads to the nozzle via groove B and channel 10 in the cylinder. FIG. B shows the rotor rotated to another position, in which groove B is disconnected from channel 3 and channel 10 in the cylinder.

The fluid treatment cartridge of the present invention can have a carrier in the channel switching means or in the middle of the channel between the liquid container and the channel switching means. The carrier in the flow switching cartridge can form the internal structure inside the rotor, and the nozzle tips extending from the cylinder can be positioned near the opposing surfaces of the carrier. Any carrier can be used as long as it can trap the nucleic acid in the sample and elute the trapped nucleic acid. The carrier can be, for example, a glass filter, a normal-phase column, an affinity resin such as a reverse-phase column, a gel filtration resin, or the like. However, it is not intended to be limited to these.

Internals within the rotor may include a liquid receiver on the exterior side of the cylinder. A space can be provided between the carrier that constitutes the internal structure and the liquid receiving portion. The liquid receiver can have a groove that is a communicable discharge channel out of the rotor from the liquid receiver. The liquid receiver can be funnel-shaped. In view A of FIG. 4 , the liquid receiver connects with the groove and groove C, and the groove C connects with the channel 4 . FIG. B shows the rotor rotated to another position, in which groove C is disconnected from channel 4 and grooves in the cylinder.

In the state of A in FIG. 4, when the cartridge is used, the cartridge is installed so that the nozzle is above the axis of gravity (vertical direction in the plane of FIG. 4), and fluid processing is performed. The liquid supplied from the flow path 3 flows out from the tip of the nozzle, the flowed-out liquid falls on the carrier, passes through the carrier, reaches the liquid receiving portion, and flows from the liquid receiving portion through the groove and the through hole A. Outflow to path 4. At the end of the flow path 4 is a negative pressure waste liquid recovery tank (not shown), and the upper space of the rotor containing the nozzle and the space between the liquid receiving part and the carrier are closed spaces, and the waste liquid recovery tank is closed. , the liquid moves toward the waste liquid collection tank.

In FIG. 5, the horizontal cross-sectional schematic explanatory drawing of an example of a flow-path switching means is shown. FIG. C shows the groove downstream of the liquid receiving portion, the through hole A and the channel 4, and these are in a state of communication. FIG. 5C shows a state in which the grooves, the through holes A, the vertical grooves C and the channels 4 shown in FIG. 4 are connected. FIG. 5D shows a state in which the rotor is rotated, the positions of the openings of the flow passages 4 in the cylinder and the through holes A in the rotor are not aligned, and the communication between the flow passages 4 and the grooves is cut off.

6 to 10 show schematic horizontal cross-sectional views and vertical cross-sectional schematic views of an example of flow switching means in which the rotor is a solid cylinder. FIG. 6 shows a through-hole B that traverses the rotor, which is a solid cylinder, and channels 20 and 21 that can connect with the through-hole B by rotating. In FIG. A, the through hole B and the flow paths 20 and 21 are in communication, and in FIG. B, the rotor rotates from the state of FIG. A, and the through hole B and the flow paths 20 and 21 are out of communication.

FIG. 7 shows a through hole C branching across the rotor, which is a solid cylinder, a groove D at one end of the through hole C, and channels 22-24 that can connect to the through hole C or groove D. . In FIG. A, the channel 22 communicates with the groove D, and the through hole C communicates with the channel 23 . In FIG. B, the rotor rotates from the state of FIG. A, the channel 22 and the groove D maintain communication, and the through hole C and the channel 24 communicate. In FIG. C, the rotor rotates from the state in FIG. B, and the through hole C does not communicate with any of the channels 22-24.

Figures 8 to 9-2 show examples of parallel movement of the rotor in the cylinder. FIG. 8 shows a through-hole D that traverses the rotor, which is a solid cylinder, and channels 25 and 26 that can be connected to the through-hole D by translation (linear motion) of the rotor. In FIG. A, the through hole D communicates with the flow paths 25 and 26. In FIG. B, the rotor moves parallel from the state of FIG. A, and the through hole D and the flow paths 25 and 26 do not communicate.

FIG. 9-1 shows a through hole E branching across the rotor, which is a solid cylinder, a groove E at one end of the through hole E, and channels 27 to 29 that can be connected to the through hole E or groove E. indicates In FIG. A, the through-hole E does not communicate with any of the channels 27-29. In FIG. B, the rotor has moved parallel from the state in FIG. In FIG. C, the rotor is moved further in parallel from the state in FIG. FIG. 9-2 is an example similar to FIG. 9-1, and FIG. D shows that the rotor is further translated from the state of FIG. are in communication with each other. FIG. E shows another embodiment of FIG. D. For example, the rotor rotates from the state shown in FIG.

In the fluid processing cartridge of the present invention, the positions of the grooves and through-holes of the rotor and the openings of the flow channels of the cylinder shown in FIGS. , the flow path to be communicated is determined, and then the liquid can be circulated between the plurality of liquid storage tanks to proceed with the desired process. Rotation or translation of the rotor can be achieved by manipulating the rotor from the outside of the cylinder.

The fluid processing cartridge of the present invention can further have a waste liquid recovery tank connected to the channel switching means via a channel, and can have an exhaust port in the waste liquid recovery tank. In FIG. 1 , the waste liquid recovery tank is indicated by 30 and has an exhaust port 31 within the waste liquid recovery tank 30 . The waste liquid recovery tank 30 is connected to the channel switching means 10 via the channel 59 . The exhaust port 31 is connected to an exhaust cartridge, such as a suction pump, provided outside the fluid processing cartridge to set the pressure in the waste liquid collection tank 30 to a negative pressure. The liquid or gas can be introduced to the waste liquid recovery tank 30 by making the inside of the liquid storage tank connected to the , and the inside of the reaction tank described later negative pressure. A plurality of liquid storage tanks and reaction tanks are used by selecting a liquid storage tank communicating with the waste liquid collection tank 30 using an exhaust cartridge, and selecting a liquid storage tank communicating with the waste liquid collection tank 30 using a flow path switching means. Liquid processing, including transfer of liquids and gases, can be easily and conveniently performed. One of the features of the cartridge of the present invention is that the liquid and gas are transferred by utilizing the negative pressure generated by the exhaust from the exhaust port provided in the waste liquid recovery tank 30 . When the cartridge is used, the cartridge is arranged such that the exhaust port is located above the waste liquid tank inlet in the waste liquid recovery tank. This is to prevent liquid leakage from the exhaust port.

The fluid processing cartridge of the present invention can further have a reaction vessel 40 connected to the channel switching means via a channel. The reaction vessel stores a nucleic acid-containing liquid treated with the cartridge of the present invention, and is used for amplifying the nucleic acid and detecting the amplified nucleic acid.

At least some of the plurality of liquid storage vessels and the reaction vessel may have vents or vent passages leading to the vents. In the embodiment shown in FIG. 1, any one of the liquid storage tanks 20 to 25 or all of the liquid storage tanks and the reaction tank 40 have vents or vent paths leading to the vents, although not shown. A liquid storage tank having a ventilation hole or a ventilation path leading to the ventilation hole is connected to the waste liquid recovery tank 30 and the flow path switching means 10 via the flow path 59, so that the inside of the tank is made negative pressure and the waste liquid recovery tank 30 is discharged. can easily lead liquids or gases to The reaction tank 40 having a ventilation hole or a ventilation path leading to the ventilation hole is connected to the flow path switching means 10 via the waste liquid recovery tank 30 and the flow path 59, so that the inside of the tank is made negative pressure and the reaction tank 40 Liquids or gases can be easily conducted. Alternatively, the reaction tank 40 may have an exhaust port similar to the exhaust port provided in the waste liquid collection tank 30, although not shown. Fluid and/or gas may be transferred to the reaction vessel 40 by exhausting air from this exhaust port to make the inside of the reaction vessel 40 negative pressure.

The fluid treatment cartridge of the present invention has the number of grooves and through-holes corresponding to the number of liquid storage tanks and the presence or absence of waste liquid recovery tanks and carriers.

The configuration and operation of the fluid processing cartridge of the present invention having a filter as a carrier inside the rotor will be further described below as an example. The cartridge of this embodiment has, as a plurality of liquid storage tanks, one sample suspension tank, two washing liquid tanks, one elution liquid tank, one reaction liquid tank, one mixing tank, and one waste liquid tank. It has a tank and one reaction tank.

Step 1 (supporting step)
Liquid: specimen suspension tank→flow switching means (nozzle→filter)→waste liquid tank Gas: specimen suspension tank vent → specimen suspension tank In step 1, the rotor of the flow path switching means moves from the specimen suspension tank. , communicates with the flow channel (corresponding to the flow channel 10 in FIG. 4) in the cylinder and the nozzle through the groove of the flow channel switching means. This channel connection is the arrangement from the channel 3 of FIG. 4A to the nozzle. Furthermore, this flow path connection, in which the through hole communicating with the waste liquid tank from below the liquid receiving portion and the flow path communicate with each other via the groove, corresponds to the arrangement of FIGS. 4A and 5C. The inside of the waste liquid tank has a negative pressure, and the liquid flows from the specimen suspension tank through the flow path into the flow path switching means, passes through the filter in the flow path switching means, and flows through the flow path switching means and the waste liquid tank. and the negative pressure waste liquid tank. If the liquid from the specimen suspension tank contains nucleic acids, the filter will trap the nucleic acids.

Step 2 (washing step)
Liquid: cleaning liquid tank→flow switching means (nozzle→filter)→waste liquid tank Gas: ventilation hole of cleaning liquid tank→cleaning liquid tank Via the groove of the path switching means, it communicates with the flow path (corresponding to the flow path 10 in FIG. 4) and the nozzle in the cylinder, and communicates with the through hole that communicates with the waste liquid tank from below the liquid receiving part. take a position to In this flow channel connection, since the flow channel connected to the groove of the flow channel switching means is the flow channel from the cleaning liquid tank, the height of the flow channel and the groove or the position on the outer circumference are different from those in step 1 (in step 1, The flow path connected to the groove of the flow path switching means is the flow path from the specimen suspension tank) is the same as the arrangement shown in FIGS. 4A and 5C. As a result, the cleaning liquid in the cleaning liquid tank flows from the cleaning liquid tank through the flow path into the nozzle in the flow path switching means, passes through the filter from the nozzle, and flows from below the liquid receiving portion to the waste liquid tank. It reaches the negative pressure waste liquid tank through the passage. Washing liquid from the washing liquid bath cleans the filter.

When two cleaning liquid tanks are used, the above operation can be repeated using two different cleaning liquid tanks for more thorough cleaning. 4A and 5C, however, the height of the flow path and the groove or the position on the outer circumference of the flow path and the groove are different from those of the first steps 1 and 2. FIG.

Step 3 (drying step)
Gas: Ventilation hole of cleaning liquid tank→Flow switching means (nozzle→filter)→Waste liquid tank Outside air is taken in from the ventilation hole of the above, and the outside air passes through the filter in the channel switching means and reaches the negative pressure waste liquid tank. This dries the filter.

Step 4 (elution step)
Liquid: eluent tank → channel switching means (nozzle → filter) → mixing tank Gas: mixing tank → channel switching means → waste liquid tank In step 4, the rotor of the channel switching means rotates, and the channel from the eluent tank is positioned to communicate with the flow channel in the cylinder (corresponding to the flow channel 10 in FIG. 4) and above the nozzle through the groove of the flow channel switching means, and the opening from below the liquid receiving part and the mixing tank take a position in which the through hole communicating with the This channel connection differs from steps 1 to 3 in the height of the channels and grooves or their positions on the circumference, but is in the mode shown in FIGS. 4A and 5C. Furthermore, in this step, the gas also flows from the mixing tank to the waste liquid tank via the channel switching means. This is the position where the channel from the mixing tank communicates with the groove of the rotor, and the groove communicates with the channel leading to the waste liquid tank. This flow path connection corresponds to the state shown in FIG. 2A. As a result, the eluent in the eluent tank flows from the eluent tank through the channel into the nozzle, passes through the filter, and reaches the mixing tank. The eluent from the eluent bath eluates and washes the nucleic acids trapped in the filter. Furthermore, the gas is sucked from the mixing tank into the negative pressure waste liquid tank via the channel switching means.

Step 5 (reaction liquid supply step)
Liquid: Reaction liquid tank → Flow switching means → Mixing tank Gas: Mixing tank → Flow switching means → Waste liquid tank In step 5, the rotor of the flow switching means rotates, and the flow path from the reaction liquid tank changes to the flow switching means. and the groove of the channel switching means communicates with the channel leading to the mixing tank. This flow path connection corresponds to the state shown in FIG. 2A. Furthermore, this positioning is the position where the channel from the mixing tank, which is the gas channel, communicates with the groove of the channel switching means, and the position where the same groove of the channel switching means communicates with the waste liquid tank. It is also positioned to communicate with the road. This channel connection also corresponds to the state shown in FIG. 2A. As a result, the reaction liquid in the reaction liquid tank reaches the mixing tank from the reaction liquid tank through the channel and the channel switching means. The eluent, reaction solution and eluted nucleic acid are mixed in the mixing tank. Further, the gas is sucked from the mixing tank into the negative pressure waste liquid tank via the channel switching means.

Step 6 (reaction tank filling step)
Liquid: Mixing tank → Channel switching means → Reaction tank Gas: Reaction tank → Channel switching means → Waste liquid tank In step 6, the rotor of the channel switching means rotates, and the channel from the mixing tank becomes the groove of the channel switching means. , and the groove of the channel switching means communicates with the channel leading to the reaction vessel. This flow path connection corresponds to the state shown in FIG. 2A. Furthermore, this positioning is a position where the flow path from the exhaust port of the reaction tank, which is the gas flow path, communicates with the groove of the flow path switching means, and the same groove of the flow path switching means communicates with the waste liquid tank. It is also a positioning that communicates with the flow path. This channel connection corresponds to the state shown in FIG. 3A. As a result, the reaction liquid in the mixing tank reaches the reaction tank via the groove of the flow path switching means from the mixed liquid tank through the flow path. The mixed reaction liquid from the mixing tank is transferred to the reaction tank. Furthermore, the gas is sucked from the reaction tank into the negative pressure waste liquid tank via the channel switching means.

The mixed reaction solution transferred to the reaction vessel is then subjected to a step of amplifying nucleic acids in the solution.

The channel switching means has a groove and through-hole structure in which the positional relationship between the rotor and the cylinder has at least a position where the reaction vessel is closed, or all channels are in a disconnected state via the channel switching means. and position. After the mixed reaction liquid is transferred to the reaction vessel, at least the reaction vessel can be closed, or all the channels can be brought into a non-connected state via the channel switching means. After that, the nucleic acid in the liquid is subjected to a step of amplification. By closing at least the reaction chamber or disconnecting all the channels, nucleic acid amplification can be performed while avoiding contamination of the reaction chamber, and further detection can be performed thereafter.

The cartridge of the present invention can be used for recovery, amplification and/or detection of nucleic acids from specimens.

<Nucleic acid amplification method>
The present invention includes a method of amplifying a nucleic acid of interest in a specimen using the fluid processing cartridge of the present invention described above, and the method can also include detection of the amplified nucleic acid in the nucleic acid amplification.

The nucleic acid to be amplified can be, for example, RNA or DNA, without any particular limitation. The nucleic acid amplification reaction can be an isothermal or thermocycling amplification reaction of nucleic acids. The isothermal amplification reaction is, for example, the LAMP method or the SmartAmp method. A thermocycling amplification reaction can be a PCR amplification reaction.

The nucleic acid amplification enzyme is not particularly limited, but may be, for example, a nucleic acid isothermal amplification reaction enzyme or a thermocycle amplification reaction enzyme. The isothermal amplification reaction of nucleic acids can be, for example, the LAMP method or the SmartAmp method of nucleic acids, and can be an enzyme for nucleic acid amplification reaction using strand displacement reaction.

Known enzymes can be used as the polymerase, which is a nucleic acid amplification reaction enzyme having strand displacement activity. For example, polymerases described in WO 2004/040019 can be mentioned, but are not intended to be limited thereto. The polymerase with strand displacement activity can also be a DNA polymerase (Aac), disclosed in WO2009/054510 (Japanese Patent No. 4450867).

As the polymerase used in the nucleic acid amplification reaction having strand displacement activity, any of normal-temperature, mesophilic, and heat-resistant polymerases can be suitably used. Moreover, this polymerase may be either a natural product or an artificially mutated mutant. Such polymerases include DNA polymerases. Examples of such DNA polymerases include thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as "B.st") and Bacillus caldotenax (hereinafter referred to as "B.ca"). Mutants lacking the 5' to 3' exonuclease activity of the derived DNA polymerase, the Klenow fragment of E. coli derived DNA polymerase I, and the like can be mentioned. DNA polymerases used in nucleic acid amplification reactions further include Vent DNA polymerase, Vent (Exo-) DNA polymerase, Deep Vent DNA polymerase, Deep Vent (Exo-) DNA polymerase, Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z -Taq DNA polymerase, Pfu DNA polymerase, Pfu turbo DNA polymerase, KOD DNA polymerase, 9°Nm DNA polymerase, Therminator DNA polymerase, Taq DNA polymerase and the like.

When the nucleic acid to be amplified is RNA, a reverse transcriptase can be used in addition to the DNA polymerase, or a DNA polymerase having reverse transcription activity can be used as the DNA polymerase. The reverse transcriptase is not particularly limited as long as it has cDNA synthesis activity using RNA as a template. 2RTase), Moloney murine leukemia virus-derived reverse transcriptase (MMLV RTase), and the like. Examples of DNA polymerases having reverse transcription activity include BcaBEST DNA polymerase, Bca(exo-) DNA polymerase, Tth DNA polymerase and the like.

The primer is appropriately selected according to the enzyme for nucleic acid amplification reaction. If the nucleic acid amplification reaction enzyme is a nucleic acid amplification reaction enzyme that utilizes a strand displacement reaction, see WO 2004/040019, JP 2009-171935, JP 2011-50380, etc. The described primers can be mentioned.

The method of the invention can further comprise, after the nucleic acid amplification procedure, the amplified nucleic acid being optically detected, electrically detected, or detected by surface plasmon resonance. Detection of the amplified nucleic acid can be performed using fluorogenic primers as primers and labeling the fluorogenic primers. Detection of amplified nucleic acids can be achieved using exciton effects by using exciton primers or exciton probes in the amplification reaction. A method for optically detecting nucleic acids may be a method using an intercalating dye.

In the method of the present invention, it is preferred that the nucleic acid amplification reaction is performed by the SmartAmp method or the LAMP method, and the exciton primer or exciton probe is used for label detection. Alternatively, it is preferable to carry out the nucleic acid amplification reaction by the PCR method and detect the label with exciton primers or exciton probes.

Following the nucleic acid amplification reaction, a nucleic acid melting curve can be drawn, and the properties of the amplification product, such as determination of false positives and true positives, can be determined from the melting curve.

INDUSTRIAL APPLICABILITY The present invention is useful in fields related to recovery, amplification and detection of nucleic acids from specimens.

10 flow path switching means 20-25 liquid storage tank 30 waste liquid recovery tank 31 exhaust port 40 reaction tanks 50-60 flow path

Claims (23)

A fluid processing cartridge comprising a plurality of liquid storage tanks, channel switching means, and channels between each liquid storage tank and the channel switching means,
The flow path switching means includes a rotor and a cylinder in which the rotor can slide while the outer peripheral surface and the inner peripheral surface of the rotor are maintained in close contact,
The rotor has at least one groove on its outer peripheral surface that enables communication between a plurality of flow passage ports of the cylinder by rotation or translation of the rotor, and/or The rotor has, by rotation or translation of the rotor, the cylinder. having at least one through-hole that allows communication of a plurality of flow channel ports;
Said cartridge. 2. A cartridge according to claim 1, wherein said groove has a through-hole opening from inside the rotor. The rotor is a solid cylinder or has a cylindrical outer wall having a cavity inside, and the through hole is a hole passing through the solid cylinder or a hole passing through the cylindrical outer wall, 3. A cartridge according to claim 1 or 2. 4. The cartridge according to any one of claims 1 to 3, wherein the plurality of liquid storage tanks, channels and cylinder are integrated, and the rotor can be inserted through the opening of the cylinder. 5. The rotor according to any one of claims 1 to 4, wherein both ends of said rotor are openings, said rotor having an internal space from each opening and having an internal structure separating two or more internal spaces. cartridge described in . The cylinder has a closed end on the inner side of the cylinder, or has a nozzle extending from the inner side of the cylinder to the inner space of the rotor, is closed except for the nozzle, has an opening at the tip of the nozzle, and the nozzle is the cylinder 6. The cartridge according to any one of claims 1 to 5, which is connectable to the channel of the . 7. The cartridge according to any one of claims 1 to 6, wherein said fluid processing cartridge has a carrier in the channel switching means or in the middle of the channel between the liquid storage tank and the channel switching means. When both ends of the rotor are openings and the rotor has an internal structure with an internal space from each opening and separating two or more internal spaces,
8. The cartridge according to claim 7, wherein the carrier in the channel switching means is included in the internal structure inside the rotor. When both ends of the rotor are openings and the rotor has an internal structure with an internal space from each opening and separating two or more internal spaces,
9. The cartridge according to any one of claims 5 to 8, wherein the internal structure includes a liquid receiving portion on the outside of the cylinder, and the liquid receiving portion has a discharge passage communicating with the outside of the rotor. 10. The cartridge of claim 9, wherein the liquid receiver is funnel-shaped. 11. The fluid processing cartridge according to any one of claims 1 to 10, further comprising a waste liquid recovery tank connected to the channel switching means via a channel, and having an exhaust port in the waste liquid recovery tank. cartridge. 12. The cartridge of claim 11, wherein the exhaust port is located above the waste tank inlet in the waste collection tank when the fluid processing cartridge is in use. 13. The cartridge according to claim 11 or 12, wherein when said fluid processing cartridge is used, the liquid in the liquid storage tank and the flow path is circulated and exhausted by negative pressure generated by the exhaust from said exhaust port. 14. The cartridge according to any one of claims 1 to 13, wherein said fluid processing cartridge further comprises a reaction tank connected to said channel switching means via a channel. At least some of the plurality of liquid storage tanks and the reaction tank have ventilation holes or air passages leading to the ventilation holes, and when the fluid processing cartridge is used, the communication port to the ventilation holes of the liquid storage tanks is the liquid storage tank. A cartridge according to any one of claims 1 to 14 located above. 16. The cartridge according to any one of claims 1 to 15, wherein the positional relationship between the rotor and the cylinder in the channel switching means has at least a position where the reaction chamber is closed. Any one of claims 1 to 16, wherein in the channel switching means, the positional relationship between the rotor and the cylinder has a structure and position of grooves and through holes in which all channels are disconnected via the channel switching means. 1. The cartridge according to item 1. The cartridge according to any one of claims 1 to 17, wherein said rotor has one or more grooves and one or more through holes. The plurality of liquid storage tanks are one or two or more sample suspension tanks, one or two or more washing liquid tanks, one or two or more eluent tanks, one or two or more mixing tanks, A cartridge according to any preceding claim, wherein the fluid processing cartridge further comprises one or more waste reservoirs and one or more reaction reservoirs. A cartridge according to any one of claims 1 to 19, wherein said cartridge is for recovery, amplification and/or detection of nucleic acids from a specimen, and nucleic acid amplification is performed in a reaction vessel. A method comprising recovery and amplification of nucleic acids using the cartridge of any one of claims 1-20. 3. When nucleic acid amplification is carried out in a reaction vessel, the rotor and the cylinder of the channel switching means are at least in a position in which the reaction vessel is closed, or in a position in which all the channels are disconnected. 21. The method according to 21. 23. The method of claim 21 or 22, further comprising the step of optically detecting, electrically detecting, or detecting by surface plasmon resonance after the nucleic acid amplification operation the amplified nucleic acid.

Images